PROJECT SUMMARY A team of investigators will develop and implement a novel, cell-based assay to discover small molecules that disrupt the attachment of virulence factors to the Gram-positive bacterial cell wall. The assay will be used to discover protein display inhibitors that target Staphylococcus aureus, a leading cause of lethal hospital- and community-acquired infections in the United States. During infections, S. aureus uses surface-attached virulence factors to adhere to specific organ tissues, resist phagocytosis, and acquire essential nutrients. These proteins are attached to the cell surface by the sortase A (SrtA) enzyme, which catalyzes a transpeptidation reaction that joins surface proteins to the cell wall. Small molecules that inhibit the enzymatic activity of SrtA could serve as powerful anti-infective agents by rendering S. aureus defenseless against the immune response. Moreover, these molecules could have broad-spectrum activity against other Gram-positive pathogens, which also rely on sortase enzymes to display virulence factors. All previously reported high- throughput screening (HTS) efforts to discover a SrtA inhibitor have employed an in vitro assay in which the enzyme exhibits limited enzymatic activity presumably because it is removed from its natural context on the cell surface where it normally associates with components of the protein secretion and cell wall synthesis machinery. As a result, most previously described SrtA inhibitors are ineffective at limiting protein display in intact cells and have, therefore, not entered clinical development. To overcome this problem, we will develop a robust, cell-based assay for HTS. The assay exploits the recently discovered growth dependence of Actinomyces oris on the activity of its SrtA enzyme, which enables sortase-specific protein display inhibitors to be identified by comparing the growth effects of small molecules on wild-type and ?srtA bacterial strains. This robust phenotype is much better suited for HTS as compared to existing low-throughput approaches that monitor SrtA activity indirectly by tracking the appearance of SrtA-attached surface proteins. The results from a preliminary pilot screen using 384-well plates and validation experiments indicate that the new cell-based assay detects S. aureus SrtA inhibitors. The approach promises to uncover potent surface display inhibitors with novel molecular scaffolds that are uniquely suited for traversing the cell wall and inhibiting SrtA enzymes located in the cell membrane.